Cancer is an aberrant net accumulation of atypical cells, which can result from an excess of proliferation, an insufficiency of cell death, or a combination of the two.
Proliferation is the culmination of a cell's progression through the cell cycle resulting in the division of one cell into two cells. The five major phases of the cell cycle are G0, G1, S, G2, and M. During the G0, phase, cells are quiescent. Most cells in the body, at one time, are in this stage. During the G1phase, cells, responding to signals to divide, produce the RNA and the proteins necessary for DNA synthesis. During the S-phase (SE, early S-phase; SM, middle S-phase; and SL, late S-phase) the cells replicate their DNA. During the G2 phase, proteins are elaborated in preparation for cell division. During the mitotic (M) phase, the cell divides into two daughter cells. Alterations in cell cycle progression occur in all cancers and may result from over-expression of genes, mutation of regulatory genes, or abrogation of DNA damage checkpoints (Hochhauser D., Anti-Cancer Chemotherapeutic Agents, 8:903, 1997).
Apoptosis or programmed cell death is the physiological process for the killing and removal of unwanted cells and the mechanism whereby chemotherapeutic agents kill cancer cells. Apoptosis is characterized by distinctive morphological changes within cells that include condensation of nuclear chromatin, cell shrinkage, nuclear disintegration, plasma membrane blebbing, and the formation of membrane-bound apoptotic bodies (Wyllie et al., Int. Rev. Cytol., 68: 251, 1980). The translocation of phosphatidylserine from the inner face of the plasma membrane to the outer face coincides with chromatin condensation and is regarded as a cellular hallmark of apoptosis (Koopman, G. et al., Blood, 84:1415, 1994). The actual mechanism of apoptosis is known to be mediated by the activation of a family of cysteine proteases, known as caspases. However, most prior art anti-cancer therapies are directed to induction of apoptosis, have proven to be less than adequate for clinical applications. Many of these therapies are inefficient or toxic, have adverse side effects, result in development of drug resistance or immunosensitization, and are debilitating for the recipient. Many diseases or conditions are characterized by undesired cellular proliferation and are know to one of ordinary skill in the medical or veterinary arts.
Induction of programmed cell death via the induction of senescence (Dimri et al., Proc. Natl. Acad. Sci USA 92:20, 1995) or apoptosis (Wyllie et al., Int. Rev. Cytol. 68:251, 1980) is important for the treatment of disorders that involve aberrant accumulation of unwanted cells such as, but not limited to, cancer, autoreactive, autoimmune, inflammatory and proliferative disorders. However, most prior art anti-cancer therapies, whether directed to induction of apoptosis or to stimulation of the immune system, have proven to be less than adequate for clinical applications. Many of these therapies are inefficient or toxic, have adverse side effects, result in development of drug resistance or immunosensitization, and are debilitating for the recipient. New methods are needed for evaluating molecules to predict whether they will possess a desired biological activity.
Synthetic oligonucleotides are polyanionic sequences that are internalized in cells (Vlassov et al., Biochim. Biophys. Acta, 11197:95, 1994). Synthetic oligonucleotides are reported that bind selectively to nucleic acids (Wagner, R., Nature, 372:333, 1994), to specific cellular proteins (Bates et al., J. Biol. Chem., 274:26369, 1999) and to specific nuclear proteins (Scaggiante et al., Eur. J. Biochem, 252:207, 1998) in order to inhibit proliferation of cancer cells.
Synthetic 27 base sequences containing guanine (G) and variable amounts of thymine (T) (oligonucleotides GTn) wherein n is ≧1 or ≦7 and wherein the number of bases is ≧20 (Scaggiante et al., Eur. J. Biochem., 252:207, 1998), are reported to inhibit growth of cancer cell lines by sequence specific binding to a 45 kDa nuclear protein, whereas GTn, wherein the number of bases is ≦20, are reported to be inactive against cancer cell lines (Morassutti et al., Nucleosides and Nucleotides, 18:1711, 1999). Two synthetic GT-rich oligonucleotides of 15 and 29 bases with 3′ aminoalkyl modifications are reported to form G-quartets that bind to nucleolin and to inhibit proliferation of cancer cell lines (Bates et al., J. Biol. Chem., 274:26369, 1999). The synthetic six base TTAGGG-phosphorothioate, having a sequence identical to that of the mammalian telomere repeat sequence, is reported to inhibit proliferation of Burkitt's lymphoma cells in vitro and in vivo (Mata et al., Toxicol. Applied Pharmacol., 144:189, 1997). However, the synthetic six base TTAGGG-phosphodiester nucleotide is reported to have no anti-telomerase activity (U.S. Pat. No. 5,643,890).
Deoxyribonucleotides with biological activity such as antisense DNA (mRNA binding or triplex-forming DNA) or immunostimulatory CpG motifs are characterized by sequence-specific linear motifs, often stabilized by intramolecular base-pair bonding. Backbone modification, such as phosphorothioate substitution, does not adversely affect and often enhances the activity of these molecules.
We have previously described a composition and method comprising 2 to 20 base 3′-OH, 5′-OH synthetic oligonucleotides selected from the group consisting of (GxTy)n, (TyGx)n, a(GxTy)n, a(TyGx)n, (GxTy)nb, (TyGx)nb, a(GxTy)nb, a(TyGx)nb, wherein x and y is an integer between 1 and 7, n is an integer between 1 and 12, a and b are one or more As, Cs, Gs or Ts, wherein the sequence is between 2 and 20 bases and wherein the sequence induces a response selected from the group consisting of induction of cell cycle arrest, inhibition of proliferation, induction of caspase activation and induction of apoptosis in a number of cancer cells (PCT CA00/01467, WO 01/44465).
Computational procedures allow a correlation of three dimensional molecular structures with biological activity, and facilitate prediction of the conformation of active molecules. Modeling entails the use of mathematical equations that are capable of representing accurately the phenomenon under study. Molecular mechanics analysis (Allinger, N. L., J. Comput. Chem., 12, 844,1991) can be used to analyze structural and conformational relationships. The fundamental assumption of molecular mechanics is that data determined experimentally for small molecules (bond length, bond angles, etc.) can be extrapolated to larger molecules. Molecular modeling approaches have been used to determine structure activity relationships and to enable the prediction of active three dimensional molecular conformations (N. Evrard-Todeschi et al., J. Chem. Inf. Comput. Sci., 38:742,1998; Chen H. et al., J. Med. Chem. 36:4094, 1993; A. Guama et al., J. Med. Chem., 40:3466,1997; M. Read, et al. Proc. Natl. Acad. Sci. USA, 98:4844, 2001).
Therefore, there is a continuing need for the identification of novel 3-dimensional conformations or structural motifs in oligonucleotides that are useful in predicting their biological activity, particularly with regard to their capability to induce cellular responses in cells. What is needed is the ability to predict cellular responses including responses such as apoptosis in cancer cells.